In cellular biology, there are many compelling questions involving processes that take place directly at the cell surface or plasma membrane. Cell-substrate interactions including attachment, spreading, morphology changes, and migration require a complex series of events to occur in a regulated and integrated manner. Cell migration, for example, plays an important role in numerous physiological and pathological processes, such as morphogenesis, wound healing, and tumor metastasis. It has been known to involve a number of coordinated events, including the protrusion of pseudopodia, the formation of new adhesions, the development of traction, and the release of old adhesions. To date, the primary tools for imaging and studying these processes have been total internal reflection fluorescence microscopy (TIRFM) and interference reflection microscopy (IRM).
Dennis Gabor invented holography in 1948 while attempting to improve the resolution of electron microscopy. Invention of the laser and the introduction of off-axis holography in the early 1960's provided the critical elements to make holography a practical and powerful tool in many areas, including microscopy, metrology, data storage, and optical processing (P. Harihan, Optical Holography, 2d Ed. (Cambridge U. Press, 2004)). In 1967, J. Goodman demonstrated the feasibility of numerical reconstruction of a holographic image using a densitometer-scanned holographic plate (J. W. Goodman, R. W. Lawrence, Digital Image Formation from Electronically Detected Holograms, Appl. Phy. Lett., 11, 77-79 (1967)).
In the IRM technique, light waves reflected from the two surfaces at the cell-substrate interface produce interference fringes, thus allowing estimation of interface thickness profile. However, here the interference image of the interface is usually complicated by the reflection image of the cell body and its contents, allowing only a qualitative interpretation of the surface profile.
In 1981, Axelrod (P. Harihan, Optical Holography, 2d Ed. (Cambridge U. Press, 2004)) demonstrated total internal reflection fluorescence microscopy (TIRFM) was effective in studying cell-substrate contacts, and it now enjoys wide employment in the biomedical industry. TIRFM uses a higher-index medium n1, and a lower-index medium n2 to reflect incident light back into the first medium. The light does not propagate into the second medium, except for an evanescent wave field, whose amplitude decays exponentially over a distance a fraction of a wavelength (˜λ/3). This evanescent wave field excites the fluorophores in TIRFM, and the penetration distance is determined by the wavelength of the incident light signal and the indices of refraction. Therefore, the sensitivity depth is adjustable by varying these parameters subject to physical constraints such as sample opacity and occlusion. Magnification of a hologram in real space holography is mostly impractical. In TIRFM, fluorophores within ˜100 nm of the coverslip's surface are excited by the evanescent field of total internal reflection, thus providing a very thin optical sectioning effect at the interface coupled with a dramatic increase in signal-to-noise ratio (SNR). Together with the exquisite specificity of GFP (green fluorescent proteins), TIRFM is used for imaging protein dynamics in calcium channels, neurotransmitters, focal adhesion, and other cellular membrane processes. On the other hand, information on the morphology of the cellular membrane surface is largely absent in TIRFM. Schnars and Jueptner, in 1994, were the first to use a CCD camera connected to a computer to record a holographic image, completely eliminating the photochemical process, in what is now referred to as digital holography (U. Schnars, Direct Phase Determination in Hologram Interferometery with Use of Digitally Recorded Holograms, J. Opt. Soc. Am., A 11, 2011-5 (1994)). Since then, developments of digital holographic techniques and applications have been gaining pace ever more rapidly. In digital holography (J. W. Goodman, R. W. Lawrence, Digital Image Formation from Electronically Detected Holograms, Appl. Phy. Lett., 11, 77-79 (1967)), the holographic interference between the object and reference fields is created optically and recorded electronically by a CCD camera. The propagation of optical field is completely and accurately described by diffraction theory, which allows numerical reconstruction of the image as an array of complex numbers representing the amplitude and phase of the optical field (J. W. Goodman, R. W. Lawrence, Digital Image Formation from Electronically Detected Holograms, Appl. Phy. Lett., 11, 77-79 (1967)). Reconstruction of the object image is carried out numerically inside a computer as an array of complex numbers. Numerical reconstruction of holographic images allows numerous imaging and processing techniques that are difficult or impossible to implement in real-space holography. A number of different methods have been considered for numerical reconstruction including Fresnel transform, Huygens convolution, and angular spectrum analysis (S. Grilli, et al., Whole Optical Wavefields Reconstructed by Digital Holography, Opt. Express 9, 294-302 (2001)). Additionally, special techniques have been developed to enhance the capabilities and to extend the range of applications. Phase-shifting digital holography allows elimination of zero-order and twin-image components even in an on-axis arrangement (P. Harihan, Optical Holography, 2d Ed. (Cambridge U. Press, 2004)). Optical scanning holography can generate holographic images of fluorescence (T. C. Poon, Three-Dimensional Image Processing and Optical Scanning Holography, Adv. Imaging & Electon Phys. 126, 329-350 (2003)). Three-channel color digital holography has also been demonstrated (I. Yamaguchi, et al., Phase Shifting ColorDigital Holography, Opt. Lett. 27, 1108 (2002)).
Optical profilers based on scanning interferometer are well suited for quantitative phase imaging applications in materials science, as in MEMS and nanofabrication (Y. Y. Cheng, J. C. Wyant, Two Wavelength Phase Shifting Interferometry, Appl. Opt. 23, 4539-43 (1984)), but the speed constraint and mechanical complexity can significantly restrict the range of applications in biology (X. Li, et al., Full Field Quantitative Phase Imaging by White-Light Interferometry with Active Phase Stabilization and its Application to Biological Samples, Opt. Lett. 31, 1830-1832 (2006)). There have been some recent developments in two-dimensional quantitative phase microscopy. In phase-shifting interference microscopy (J. Beuthan, et al., The Spatial Variation of the Refractive Index in Biological Cells, Phys. Med. Biol. 41, 369-382 (1996)), the quantitative phase image is obtained from a combination of three or more interferograms. There is also a non-interferometric method to extract quantitative phase image from differential focusing property of bright-field intensity images alone (A. Barty, et al., Quantitative Optical Phase Microscopy, Opt. Lett. 23, 817-9 (1998)).
Application of digital holography in microscopy is especially important, because of the very narrow depth of focus of high-magnification systems. Numerical focusing of holographic images can be accomplished from a single hologram. Direct accessibility of phase information can be utilized for numerical correction of various aberrations of the optical system, such as field curvature and anamorphism (P. Ferraro, et al., Compensation of the Inherent Wave Front Curvature in Digital Holographic Coherent Microscopy for Quantitative Phase-Contrast Imaging, Appl. Opt. 42, 1938-46 (2003)). Digital holography has been particularly useful in metrology, deformation measurement, and vibrational analysis (M. L. Xu, et al., Studies of Digital Microscopic Holography with Applications to Microstructure Testing, Appl. Opt. 40, 5046-5051 (2001)). Microscopic imaging by digital holography has been applied to imaging of microstructures and biological systems. Digital interference holography has been developed for optical tomographic imaging as well as multiwavelength phase contrast digital holography for high resolution microscopy.
Therefore, with the limitations of TIRFM and IRM in mind, it becomes readily apparent that a technique to generate accurate, quantitative surface profile images of live cellular membranes is needed and will greatly help us better understand the important process of cellular motion. Therefore, a quantitative method that takes advantage of the strengths of TIRFM, without suffering from its above-mentioned drawbacks, would have useful applications in surface profile characterization and the study of cellular motion. With this motivation in mind, a solution is offered by digital holographic microscopy.